In general, a drug is processed into a formulation by which pharmacological efficacy can be optimally exhibited, and is then administered into a living body through various routes. The administered drug is released from the formulation and exhibits various pharmaceutical effects in vivo while undergoing absorption, distribution, metabolism and excretion. In order for the drug to safely and effectively exhibit its pharmaceutical efficacy in vivo and to be selectively active on an intended site of the body, it is necessary to control the behavior of drug in vivo. A drug delivery system (DDS) is a formulation designed to effectively deliver an appropriately necessary amount of drug by maximizing the efficacy of drug while suppressing side effects of drug.
Although DDS has not yet been completely defined, it has been used to embrace in a broad sense the meaning of a wide range of formation designs controlling the behavior of drugs in vivo, inclusive of a targeting system and a chemical delivery system, and to embrace in a narrow sense the meaning of a controlled release system.
Controlled drug release methods have rapidly developed in pharmaceutical fields since the 1970s. A micromolecular controlled release system has been typically applied in medical fields, and it has been in particular utilized in delivering pharmaceutical products to a specific site of the human body.
A controlled release system exists in various forms including capsules for oral administration, matrices, microcapsules for oral administration and injection, microspheres, microparticles, nanoparticles, liposomes and implants (J. Kost, 1995).
Microencapsulation
Microencapsulation is the most important technique in controlled release preparation. In this field, as nano-technology is combined with the development of drug delivery systems in the pharmaceutical fields, the of microparticles has rapidly developed.
For example, experiments in which pseudophedrine HCl, which is a soluble pharmaceutic product, is entrapped in polymeric microspheres using oil-in-water (O/W) dispersion or co-solvent methods, water-in-oil-in-water (W/O/W) multiple emulsion method or an emulsion-solvent evaporation method, have demonstrated that an appropriate amount of drug can be loaded (R. Bodmeier et al., 1991). A microcapsule refers to a spherical particle having solid or liquid drug positioned at a center nucleus, and a microsphere refers to multiple nuclei in which solid or liquid drug is dispersed in a polymeric material. A microparticle is used to embrace microcapsules and microspheres, and refers to a microparticular drug carrier using microparticles such as a polymeric matrix or lipid as a drug carrier. Unless specified otherwise throughout the specification, these terms are used to those described above.
Microparticles can be produced to have various diameters ranging from 0.1 μm to several hundreds of micrometers (μm). Among those microparticles, ones having a diameter of 1 μm or less are referred to as nanospheres (or nanoparticles). Most microspheres generally preserved in a solid phase to be suspended during use have been traditionally used as radiation diagnosis reagents, and much attention has been recently paid thereto as a drug carrier.
Drug carrier
Physical supports (carriers) for use in a controlled release system include various types of synthetic and natural rubber. Among them, biodegradable polymers are albumin, gelatine, collagen, fibrinogen, polylactides (PLA), polyglycolides (PGA), poly β-hydroxy butyric acids (PHB), polycaprolactone, polyanhydrides, polyorthoesters, poly(lactic-co-glycolic) acid (PLGA), which is a copolymer of these materials, and the like. These polymers were developed in the 1960s as surgical suture threads, and various kinds of research into the polymers as systems of sustained release preparation such as steroids, anti-malaria agents, narcotic inhibitors or carcinostatis substances have been made since the 1970s. After Tice et al. disclosed that sustained release could be achieved by producing water-soluble compounds, e.g., antibiotics and luteinizing hormone-releasing hormone (LHRH) analogues, from microsphere preparations, much more attention has been being paid to the above-described biodegradable polymers (Tice, T. R. (1984) Pharm. Tech. 8, 26–36).
PLGA
In order to achieve microencapsulation of peptides with copolymers, the following requirements must be met:                1) The peptides must be formed of biodegradable polymers;        2) The denaturation of peptides must not occur during the process; and        3) Encapsulation efficiency must be sufficiently high.        
Currently, main research into microencapsulation of peptides and proteins is a water-in-oil-in-water (W/O/W) solvent evaporation technique using poly-(lactic acid) (PLA) or poly(lactic-co-glycolic) acid (PLGA) which is a PLA and poly-glycolic acid (PGA) copolymer.
The PLGA and PLA are polymers that are fully supported by toxicological and clinical data and are nontoxic, biocompatible, biodegradable polymers that are authorized by the FDA to be used for the human body. Glycolic acid and lactic acid which are denatured from these materials are removed through internal metabolism. Since the rate of hydrolysis of those polymers depends upon temperature, the presence of a catalyst and a definite change in pH, a change in the rate of denaturation depending upon the position in the body is not observed. This satisfies requirements suitable for use in drug delivery formulation. The rate of denaturation is determined by the molecular weight and crystallinity of a polymer and the lactide:glycolide ratio of the PLGA. Since the lactic acid has asymmetric carbon atoms, it has two optical isomers. Thus, the polymers consist of L, D- and D, L-lactic acid. The L, D-polymer is in a crystalline form, the D, L-polymer is in an amorphous form and these polymers are denatured rapidly (Patrick Convreur, Maria Jose, Blanco-Prieto, Francis Puisienx, Bernard Reques, Elias Fattal, Multiple emulsion technology for the design of microspheres containing peptides and oligopeptide, Advanced Drug Delivery Review 28 (1997) 85–96).
The PLGA is used in microencapsulation of water-soluble and insoluble pharmaceutical products. In the case where microspheres were produced using bovine serum albumin (BSA) as a model protein by an oil-in-oil (O/O), oil-in-water (O/W) or water-in-oil-in-water (W/O/W) emulsion method, it turned out that 50 to 70% of the overall proteins injected into a formulation medium was contained in the microspheres and the particle sizes thereof were 500 μm, 25 to 100 μm and 10 to 20 μm, respectively. The release types of BSA were different according to the method. In the case of microspheres vacuum dried after being produced by the W/O/W emulsion method, the release amount thereof was large. In the case of microspheres containing carbopol-R 951 and produced by the O/W method, the 5 initial burst amounts were large. The release lasting periods were 54 days, 36 days and 34 days, respectively.
Also, in the case of microspheres produced from PLGA by a double emulsion method, it was reported that the microspheres were produced in a size suitable for pulmonary delivery. In the case of microspheres produced using Ciliary neurotrophic fact (CNTF) and PLGA in a mixture ratio of 30:70 (D. Maysinger, et al. 1996), it was reported through phase analysis that the average particle size of the microspheres was 1.76±0.0186 μm.
Insulin
Insulin is one of targets of controlled release preparation that is being researched most vigorously. Currently, the most typically used insulin is in the form of injection and pump. 40, 80 and 100 IU/ml of Zn-insulin suspensions or neutral solutions, which typically have immediate effects, are in widespread use, and 100 IU/ml is generally preferred in use. Insulin tends to exhibit fast efficacy in a solution state rather than in a crystal state, while having a short time in a protracted action. Thus, patients use insulin mostly in the form of injections before meals or before sleeping. According to activity, immediately active insulin and protractedly active insulin are separately administered. Such existing insulin preparations are an inconvenience in a patient's daily life due to the necessity of frequent administrations of insulin. In the case of missing an administration, a sharp decrease in the serum glucose concentration or a significant risk of hypoglycemia may occur.
It is known that insulin crystals have longer pharmaceutical efficacy than insulin solutions and are active for about 36 hours when they are administered. This is because insulin crystals slowly dissolve insulin in the body, which can be advantageously used in protracting the activity of insulin within blood, in a manner similar to that in which insulin is generated and secreted from the pancreas of the human body. On the basis of this fact, in 1956, Schlichtkrull attempted to generate insulin crystals small enough to be used for treatment of diabetes. Thereafter, in the pharmacy field, continuous research has been conducted into crystallization methods for making microcrystals of insulin or insulin derivatives so as to be slowly dissolved in the body. Many reports of insulin crystallization methods have been hitherto made, and most reports have utilized a change in the pH of insulin solution (U.S. Pat. Nos. 3,719,655 and 4,959,351). However, these conventional crystallization methods present a problem in that fine insulin crystals of 10 μm or less, which are suitable for administration through the lung, cannot be obtained at high yield.
Various researches are being conducted into controlled release preparation of insulin which is capable of reducing the concentration of serum glucose continuously for a long time after administration in vivo, and among them, several insulin controlled release preparations have shown significant developments. Most insulin delivery systems currently being researched are based on the reaction between glucose-oxidase tied up into a polymer in a drug delivery system (DDS) and glucose present in blood. If the reaction between glucose and glucos-oxidase results in a decrease in the pH in the microenvironment of the DDS, a polymer system is swollen so that the amount of released insulin increases. The polymer system used herein includes N,N-dimethylaminoehtyl methacryalte and poly-acrylamide.
Alternatively, in order to prevent insulin for oral administration from being denatured in the digestive system, nanospheres may be produced. According to this method, protein denaturation is prevented by a polymer matrix before being absorbed through intestinal M cells and the polymer matrix of a small size allows permeation through a membrane barrier. This method is advantageous in view of the convenience in administration. However, the delivery effect of this method is only 11% the effect of abdominal delivery (Gerardo P. Carino, Jules S. Jacob, Edith Mathiowitz, Nanosphere based oral insulin delivery, Journal of Controlled Release 65 (2000) 261–269).
Pulmonary delivery is one of the active researches that are currently being conducted. The pulmonary delivery provides a relatively wide surface area as large as a wide tennis court, e.g., 100 m2, compared to nasal delivery, and allows for fast absorption through a vast number of blood vessels present over thin epithelial cell walls. According to the results of clinical trials conducted by several companies, announced in a conference of the America Diabetes Association, the same effect as injected insulin could be obtained. It is known that a clinical trial phase III in which approximately 1,000 patients are the subjects of the clinical experimentation is under way. The development of a delivery system of protein pharmaceutical products using pulmonary delivery is expected to propose ways of easy and convenient administration of protein pharmaceutical products that have been conventionally supplied only through injection. The present invention provides an effective controlled release preparation of insulin in the context of pulmonary delivery of insulin.
Stability of protein pharmaceutical products
In the case of small peptides, even if decomposition thereof scarcely occurs, the stability thereof is an essential issue. In the course of producing microparticles, proteins or peptides are applied to excess stress. Thus, the microencapsulation process of protein pharmaceutical products must be free from excessive heat and shear stress, a sharp change in pH, organic solvent and excessive freezing and drying. The microencapsulated proteins may be hydrated even during storage and the proteins are prone to denaturation and aggregation under these circumstances. When the polymer begins to be decomposed after being administered, a highly concentrated acidic microenvironment is created inside and around the polymer due to decomposed acidic monomer. Under these circumstances, the proteins are prone to aggregation, hydrolysis and chemical change. Finally, the proteins may cause reversible or irreversible partition together with the polymer, thereby affecting a drug delivery rate, finally leading to denaturation, aggregation and inactivation of proteins.
Among peptides for therapeutic purposes, insulin becomes a target of a nuclease and is prone to chemical, physical denaturation in a solution or suspension (J. Brange, L. Andersen, E. D. Laursen, G. Meyn, E. Rasmussen, Toward understanding insulin fibrillation, J. Pharm. Sci. 86 (1997) 517–525).
Deamidated products may be generated in a liquid phase solution due to chemical decomposition of insulin, e.g., desamido A21 in acidic media or desamido B3 in a neutral solution. Otherwise, insulin dimers having a covalent bond may be formed by transamidation (R. T. Darrington, B. D. Anderson, Evidence for a common intermediate in insulin deamidation and covalent dimer formation; effects of pH and aniline trapping in dilute acidic solutions, J. Pharm. Sci. 84 (1995) 274–282).
Like other globular proteins, insulin has a tertiary structure in which the hydrophobic surface thereof is concealed inside through folding or assembly of various molecules. Otherwise, a change of its native conformation may be affected for various reasons. Specifically, heat, physical force and exposure to hydrophobic surface cause a structural change of proteins, thereby resulting in aggregation of proteins and producing insoluble precipitates (B. V. Fisher, P. B. Porter, Stability of bovine insulin, J. Pharm. Pharmacol. 33 (1981) 203–206).
The denaturation of protein pharmaceutical products causes immunogenicity or antigenicity, accompanied by generation of antibodies, impeding the activation of injected proteins and affecting the action of an identical protein spontaneously generated in the human body, which is very hazardous.
Therefore, consideration must be taken into the stability of pharmaceutical products in view of formulation as well as . The present invention relates to a microencapsulation method while attaining stability of insulin contained in formulations.
Disclosure of the Invention
Formulation and drug delivery are very important factors in developing protein pharmaceutical products using gene recombinant technology. In particular, since biological and pharmaceutical products have a large molecular weight and a tertiary structure, on which the activity and physical property are greatly dependent, they are easily prone to denaturation, compared to general chemical synthetic drugs. As a result, formulation becomes an important issue in view of structural stability.
Since insulin is easily denatured during microencapsulation, it is liable to generate deamindated products, and approximately 50% of a loss in the overall proteins occurs. Also, the initial burst occurring due to partition on the surface of the protein while drying microparticles, may cause hypoglycemic effects.
Therefore, it is a first object of the present invention to provide a controlled release preparation of insulin, which can minimize protein denaturation of insulin and can increase stability in the course of microencapsulation, and its method thereof.
To this end, the present invention provides a controlled release preparation of insulin, by which microparticles of insulin are microencapsulated using a polymer carrier. The controlled release preparation according to the present invention can be made in various forms suitable for pulmonary inhaled administration, injection, oral administration, transdermal suction and so on. In particular, the size of the microparticles is 10 μm or less, preferably 5 μm or less, so as to be suitably used for pulmonary delivery. The insulin controlled release preparation according to the present invention is a sustained release preparation which can reduce the number of administrations of insulin owing to continuous exhibition of pharmaceutical efficacy. Also, according to the present invention, the amount of initial burst of insulin can be controlled to prevent a sharp decrease in the serum glucose concentration.
It is another object of the present invention to provide increased encapsulation efficiency and optimization of insulin.
Biodegradable polymers used for microencapsulation differ in decomposition rate according to physical properties and compositions thereof, and a considerable time is required for complete decomposition. In the case of PLGA whose safety relative the human body has already been verified, it is generally known that approximately 50 days to approximately 100 days are required for decomposition of the PLGA even if the decomposition rate thereof may differ according to the composition. Thus, if a microencapsulated preparation using a biodegradable polymer as a carrier is continuously administered, polymer accumulation in a living body may occur. Therefore, it is an object of formulation technology to entrap a larger amount of drug using the same amount of the polymer carrier in microencapsulation.
In the case of microencapsulation of a drug in a liquid state, the concentration of the drug that can be entrapped in microparticles is determined by the solubility of the drug. If the solubility of the drug is low, only a small amount of the drug is entrapped. In this case, in order to deliver an appropriate amount of the drug, it is necessary to administer a much greater volume of microparticles into a living body.
This problem becomes serious in the case of pulmonary delivery. In general, a substance delivered through the lung is subjected to internal absorption. A non-absorptive substance is removed by a clearance process using macrophages. The clearance process takes more time in the blastosphere than in the airway where the delivered substance is removed within 24 hours (Camner, P. 1994). Also, it was reported in a paper that the clearance process is carried out more slowly when more substances exist in the lung.
Therefore, in particular, when a drug is administered through the lung, it is necessary to reduce an amount of polymeric substance to be administered into a body by increasing the ratio of the drug to the polymer carrier. In the present invention, there is provided a controlled release preparation of insulin, by which the content of insulin contained in microparticles can be adjusted as needed by producing microparticles using uniform microparticles of insulin.
It is a still another object of the present invention to provide a method of preserving microparticles of insulin in a liquid phase in order to solve the inconvenience of use of microencapsulated insulin, that is, the microencapsulated insulin should be again dispersed in a solution.
Generally, microparticles are freeze-dried for storage and dispersed prior to use. However, in the case of protein pharmaceutical products, denaturation may occur in the course of freeze-drying. Also, initial burst may occur due to surface partition of the microparticles of the drug, which is caused in the course of drying. Thus, in order to facilitate drug administration and to prevent initial burst, the present invention provides a method of preserving a microparticle preparation of insulin in a solution using an isoelectric point without freeze-drying the microparticle preparation of insulin.
Alternatively, the present invention provides a controlled release preparation of insulin and its method, by which immediate and protracted effects can be simultaneously attained at a single administration by including an immediately active portion of insulin in the preparation together with microparticles of insulin crystals.
The other objects and advantages of the present invention will be described below and will be apparent by embodiments of the present invention.